BK channels are fundamental components of neuronal signaling through effects on neuronal excitability and synaptic transmission. This proposal is aimed at determining the fundamental mechanisms that govern expression and localization of BK channels in mammalian hippocampus. Using novel, state-of- the art mass spectrometric approaches we have made great inroads in defining the in vivo phosphorylation sites on the primary or BK1 subunit of BK channels purified from rat brain. We find that most in vivo phosphorylation sites are Pro-associated pSer and conform to consensus binding sites for proteins containing pSer-binding modules. Moreover, these sites are likely phosphorylated by proline- directed kinases (ProDKs), whose importance in neuronal function and as targets for new therapeutics is just now being appreciated. These data provide the first opportunity to investigate the role of bona fide and unambiguously identified in vivo brain phosphosites on BK channels in governing their expression levels and subcellular localization in hippocampus. We will test the overall hypothesis of this proposal that these sites are crucial to neuronal function and plasticity as mediated by ProDKs acting on native BK channels. In aim 1 we will accomplish this by examining the effects of interventions that alter the phosphorylation state of BK1. We will mutate identified in vivo ProDK phosphorylation sites, and will intervene in ProDK expression levels in heterologous cells and hippocampal neurons, and determine effects on BK channel expression. In Aim 2 we will use similar approaches to determine the role of these phosphorylation sites in polarized localization of BK channels. These studies will yield important insights into the physiological and pathological regulation of BK channels, which are key regulators of neuronal excitability and synaptic transmission in mammalian hippocampus. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for neurological and psychiatric disorders.